In recent years, the mechanisms of mucosal immunity on the respiratory apparatus, the digestive apparatus, the reproductive organs, and other organs have been gradually elucidated as the immune system to prevent infectious diseases such as influenza or acquired immunodeficiency syndrome (AIDS). For example, immune response to prevent influenza virus infection is associated with mucosal IgA antibody, serum IgG antibody to neutralize the viruses, and cytotoxic T cells that lyse infected cells to interrupt virus transmission. Such mucosal immune mechanisms are functional at the initial phase of infection, and play a key role in biophylaxis at the time of infection or during the initial phase of infection. Accordingly, mucosal vaccines inducing immune protection response against infection on the mucosa, which is the first barrier at portals of entry for pathogens, are considered as effective vaccine for various infectious diseases through mucosae.
While mucosal vaccines induce secretory IgA antibody in mucosal tissue upon mucosal administration (e.g., intranasal administration), and also induce IgG antibody in the serum. Thus, mucosal vaccines are capable of inducing immune responses in both the mucosal and systemic systems against pathogens. In addition, mucosal vaccines are superior to conventional vaccination with needles and syringe in terms of operability, safety, and economic efficiency. Accordingly, mucosal vaccines are expected as novel vaccines, and have been developed.
However, because mucosal vaccines with antigens alone are not capable of inducing sufficient immune responses, mucosal adjuvants for mucosal vaccines is necessary in order to induce effective immune responses on the mucosal surface. Up to the present, many mucosal adjuvants have been reported. For example, bacterial endotoxins such as cholera toxin (CT) and heat-labile enterotoxin (LT) of enterotoxigenic Escherichia coli, have been known as representative mucosal adjuvants (Non-Patent Documents 1 and 2). However, previous reports showed that clinical trials with LT intranasal administration caused facial nerve palsy (Bell's palsy). Accordingly, development of mucosal adjuvants with toxins such as CT or LT might be difficult in terms of safety. MPL resulting from attenuation of activity of endotoxin LPS, bacterial flagellin proteins (Patent Document 1), double-stranded RNA (poly(I:C)) (Patent Document 2), and other substances have been studied as mucosal adjuvants, which are not derived from toxins. However, since those candidates induce excessive inflammatory responses, they are not satisfactory for mucosal adjuvants in terms of safety. That is, no effective and safe adjuvants for mucosal vaccines are being put to practical use at present.
The hemagglutinin (HA) and the nontoxic-nonhemagglutinin (NTNH) component bind to the botulinum neurotoxin (NTX) produced by botulinum bacilli causing food poisoning, and those components form three types of neurotoxin complex (progenitor toxin (PTX)) whose molecular weight are 300,000, 500,000, or 900,000. Botulinum toxin blocks neuron transmission, and leads to death in human. Taking advantage of the activity thereof, botulinum toxin is used as an effective neurotransmission inhibitor for medical purposes. For example, a botulinum toxin type A (BOTOX) complex is known to be used for treatment of blepharospasm, hemifacial spasm, spasmodic torticollis, heterotropia, and the reduction of wrinkles. In the neurotoxin complex as described above, non-toxic HA is known to have functions of disrupting the epithelial barrier and transporting botulinum neurotoxins and macromolecules. When NTX and albumin antigens are subcutaneously administered to mice in combination with HA, production of serum antibody specific for antigens is enhanced through IL-6 production (Non-Patent Document 3). While Patent Documents 3 and 4 describe the adjuvant activity of an HA subcomponent (HA1 or HA3) and the use as a carrier of nucleic acids into cells, no protein complex composed of HA subcomponents (HA1, HA2, and HA3) has been discussed. The present inventors previously reported that HA acts on M cells in the epithelial cell layer of the Peyer's patch (i.e., M cells on the Peyer's patch), and that HA assists migration of neurotoxin complex from apical side of to basolateral side of M cells via transcytosis (Non-Patent Document 4). While the functions of the neurotoxin complex (HA to which the toxin component has been bound) to breach the intestinal epithelial barrier have been investigated in the study described above, interaction of toxin-free HA with M cells or adjuvant effects for delivering vaccine antigens for mucosal vaccines to infectious diseases have not yet been examined.